The key aspect of the conceptual framework in the pathogenesis of aplastic anemia (AA) relates to the events that can trigger immune attacks directed against hematopoietic progenitor and stem cells. Immune tumor surveillance reactions to emerging clonal outgrowth with a cross-reactivity and collateral damage to the stem cell compartment have been proposed among various types of self-directed immune responses in AA. This possibility fits with the close pathophysiologic association of AA with clonal myelodysplastic syndromes (MDS). The latter is a common late-stage complication of AA and constitutes, in its hypocellular form, an alternative diagnosis in misdiagnosed AA because of the lack of sufficient cells for cytomorphologic or cytogenetic evaluation. An alternative theory explaining the pathophysiological relationship of AA to clonal diseases of hematopoiesis relates to the possibility that oligoclonality due to stem cell depletion may lead to a higher probability f recruitment of defective stem cells. New molecular technologies, including high-density SNP arrays and next generation sequencing (NGS), have created opportunities for study of the molecular pathogenesis of AA. These new technologies allow for unbiased screens for mutations involving major portions of the genome. They also allow for high sensitivity and analysis of clonal architecture. Our preliminary results suggest that clonal events may be detected in AA at presentation or occur very early in the course of the disease. While the presence of somatic mutations may not necessarily result in MDS in every instance, it is likely that if subsequent MDS develops it happens on the basis of these lesions. In this proposal we hypothesize that clonal events may be detected at initial presentation in a proportion of AA patients, and that certain clonal events are permissive and lead to late clonal evolution and progression through acquisition of secondary events, while others may be eradicated by the immune system. The objectives of this project are to assess the proportion of AA patients harboring somatic clonal lesions and determine the prognostic significance and diagnostic utility of such genetic defects in terms of response to immunosuppression and clonal evolution. In addition we hope to identify ancestral events in these patients that determine which secondary events lead to MDS. We will screen cohorts of patients with AA as well as post-AA clonal evolution, including MDS and PNH, for the presence of somatic clonal lesions during the course of their disease. By serial and cross-sectional analysis we will investigate hierarchical clonal architecture consisting of somatic mutations and chromosomal analysis. Finally, we will establish clinical correlations and the prognostic impact of these clonal somatic events. Clinical analyses will be performed to determine the impact of somatic mutations on risk of evolution vs. sub clonal persistence, response/refractoriness to immunosuppression, and the speed and dynamics of PNH persistence or evolution. We postulate that addressing these important questions in AA may lead to paradigm-shifting concepts in the understanding of this disease.